A two-site kinetic mechanism for ATP binding and hydrolysis by E. coli Rep helicase dimer bound to a single-stranded oligodeoxynucleotide.

Escherichia coli Rep helicase catalyzes the unwinding of duplex DNA in reactions that are coupled to ATP binding and hydrolysis. We have investigated the kinetic mechanism of ATP binding and hydrolysis by a proposed intermediate in Rep-catalyzed DNA unwinding, the Rep "P2S" dimer (formed with the single-stranded (ss) oligodeoxynucleotide, (dT)16), in which only one subunit of a Rep homo-dimer is bound to ssDNA. Pre-steady-state quenched-flow studies under both single turnover and multiple turnover conditions as well as fluorescence stopped-flow studies were used (4 degrees C, pH 7.5, 6 mM NaCl, 5 mM MgCl2, 10 % (v/v) glycerol). Although steady-state studies indicate that a single ATPase site dominates the kinetics (kcat=17(+/-2) s-1; KM=3 microM), pre-steady-state studies provide evidence for a two-ATP site mechanism in which both sites of the dimer are catalytically active and communicate allosterically. Single turnover ATPase studies indicate that ATP hydrolysis does not require the simultaneous binding of two ATP molecules, and under these conditions release of product (ADP-Pi) is preceded by a slow rate-limiting isomerization ( approximately 0.2 s-1). However, product (ADP or Pi) release is not rate-limiting under multiple turnover conditions, indicating the involvement of a second ATP site under conditions of excess ATP. Stopped-flow fluorescence studies monitoring ATP-induced changes in Rep's tryptophan fluorescence displayed biphasic time courses. The binding of the first ATP occurs by a two-step mechanism in which binding (k+1=1.5(+/-0.2)x10(7) M-1 s-1, k-1=29(+/-2) s-1) is followed by a protein conformational change (k+2=23(+/-3) s-1), monitored by an enhancement of Trp fluorescence. The second Trp fluorescence quenching phase is associated with binding of a second ATP. The first ATP appears to bind to the DNA-free subunit and hydrolysis induces a global conformational change to form a high energy intermediate state with tightly bound (ADP-Pi). Binding of the second ATP then leads to the steady-state ATP cycle. As proposed previously, the role of steady-state ATP hydrolysis by the DNA-bound Rep subunit may be to maintain the DNA-free subunit in an activated state in preparation for binding a second fragment of DNA as needed for translocation and/or DNA unwinding. We propose that the roles of the two ATP sites may alternate upon binding DNA to the second subunit of the Rep dimer during unwinding and translocation using a subunit switching mechanism.     

The effects of cisplatinum and vincristine on peripheral nerve regeneration

Current treatment modalities for extremity sarcoma often include tumor extirpation plus neoadjuvant therapy. Limb-sparing surgery may require reconstruction of critical nerve defects. Neurotoxic side effects from adjuvant chemotherapy have been reported and raise concerns regarding the effects of chemotherapy on nerve regeneration. In an attempt to define the effects of adjuvant chemotherapy on peripheral nerve regeneration, cisplatin and vincristine were administered to rats following isografting of the posterior tibial nerve. Parameters used to assess peripheral nerve regeneration included walking track analysis and histomorphology. Sixty 250-g Sprague-Dawley rats were randomly allocated into one of three treatment groups. Each animal underwent a 15-mm reversed interposition nerve isograft from 30 donor rats into the right posterior tibial nerve. Ten animals served as control. The remaining animals were divided into two groups of 25 animals each. One group received cisplatin (75 mg/m2) and the other group received vincristine (1 mg/m2). Chemotherapy was administered at 4-week cycles for a total of six cycles (24 weeks). Walking track analysis was performed monthly. Nerve specimens were harvested from the grafted segment and the distal posterior tibial nerve for histomorphology. Walking track analysis demonstrated no statistical difference in print length between the control and chemotherapeutic groups at the conclusion of the study. The number of axons per square millimeter and nerve fiber density were not statistically different between control and chemotherapeutic groups. In the rodent posterior tibial nerve model, postoperative adjuvant therapy does not significantly alter functional outcome in peripheral nerve regeneration. The practice of immediate nerve grafting after tumor extirpation, despite planned postoperative chemotherapy, is supported.     

Hemodynamic response and oxygen transport in pigs resuscitated with maleimide-polyethylene glycol-modified hemoglobin (MP4).

Cell-free Hb increases systemic and pulmonary pressure and resistance and reduces cardiac output and heart rate in animals and humans, effects that have limited their clinical development as "blood substitutes." The primary aim of this study was to evaluate the hemodynamic response to infusion of several formulations of a new polyethylene glycol (PEG)-modified human Hb [maleimide PEG Hb (MalPEGHb)] in swine, an animal known to be sensitive to Hb-induced vasoconstriction. Anesthetized animals underwent controlled hemorrhage (50% of blood volume), followed by resuscitation (70% of shed volume) with 10% pentastarch (PS), 4% MalPEG-Hb in lactated Ringer (MP4), 4% MalPEG-Hb in pentastarch (HS4), 2% MalPEG-Hb in pentastarch (HS2), or 4% stroma-free Hb in lactated Ringer solution (SFH). Compared with baseline, restoration of blood volume after resuscitation was similar and not significantly different for the PS (103%), HS2 (99%), HS4 (106%), and MP4 (87%) animals but significantly less for the SFH animals (66%) (P < 0.05). All solutions that contained MalPEG-Hb restored mean arterial and pulmonary pressure and cardiac output. Systemic vascular resistance was unchanged, and pulmonary arterial pressure and resistance were increased slightly. Both systemic and pulmonary vascular resistance increased significantly in animals that received SFH, despite less adequate blood volume restoration. Oxygen consumption was maintained in all animals that received MalPEG-Hb, but not PS. Base excess improved only with MalPEG-Hb and PS, but not SFH. Red blood cell O2 extraction was significantly increased in animals that received Hb, regardless of formulation. These data demonstrate resuscitation with MalPEG-human Hb without increasing systemic vascular resistance and support our previous observations in animals suggesting that the efficacy of low concentrations of PEG-Hb in the plasma results from reduced vasoconstriction.     

Kinetic mechanism for formation of the active,dimeric UvrD helicase-DNA complex

Escherichia coli UvrD protein is a 3' to 5' SF1 helicase required for DNA repair as well as DNA replication of certain plasmids. We have shown previously that UvrD can self-associate to form dimers and tetramers in the absence of DNA, but that a UvrD dimer is required to form an active helicase-DNA complex in vitro. Here we have used pre-steady state, chemical quenched flow methods to examine the kinetic mechanism for formation of the active, dimeric helicase-DNA complex. Experiments were designed to examine the steps leading to formation of the active complex, separate from the subsequent DNA unwinding steps. The results show that the active dimeric complex can form via two pathways. The first, faster path involves direct binding to the DNA substrate of a pre-assembled UvrD dimer (dimer path), whereas the second, slower path proceeds via sequential binding to the DNA substrate of two UvrD monomers (monomer path), which then assemble on the DNA to form the dimeric helicase. The rate-limiting step within the monomer pathway involves dimer assembly on the DNA. These results show that UvrD dimers that pre-assemble in the absence of DNA are intermediates along the pathway to formation of the functional dimeric UvrD helicase.     

Vacuum assisted closure for the treatment of sternal wounds: the bridge between débridement and definitive closure

A method to refine the treatment of sternal wounds using Vacuum Assisted Closure (V.A.C.) therapy as the bridge between débridement and delayed definitive closure is described. A retrospective review of 35 consecutive patients with sternal wound complications over a 2-year period (March of 1999 to March of 2001) was performed. The treatment of sternal wounds with traditional twice-a-day dressing changes was compared with the treatment with the wound V.A.C. device. An analysis of the number of days between initial débridement and closure, number of dressing changes, number and types of flaps needed for reconstruction, and complications was performed. Eighteen patients were treated with traditional twice-a-day dressing changes and 17 patients were treated with V.A.C. therapy alone. The two groups were similar regarding age, sex, type of cardiac procedure, and type of sternal wound. The V.A.C. therapy group had a trend toward a shorter interval between débridement and closure, with a mean of 6.2 days, whereas the dressing change group had mean of 8.5 days. The V.A.C. therapy group had a significantly lower number of dressing changes, with a mean of three, whereas the twice-a-day dressing change group had a mean of 17 (p < 0.05). Reconstruction required an average of 1.5 soft-tissue flaps per patient treated with traditional dressing changes versus 0.9 soft-tissue flaps per patient for those treated with V.A.C. therapy (p < 0.05). Before closure, there was one death among patients undergoing dressing changes and three in the V.A.C. therapy group, all of which were unrelated to the management of the sternal wound. Patients with sternal wounds who have benefited from V.A.C. therapy alone have a significant decrease in the number of dressing changes and number of soft-tissue flaps needed for closure. Finally, the V.A.C. therapy group had a trend toward a decreased number of days between débridement and closure.     

Evolution of spore release mechanisms in the saprolegniaceae (Oomycetes): evidence from a phylogenetic analysis of internal transcribed spacer sequences

Classical studies on spore release within the Saprolegniaceae (Oomycetes) led to the proposition that different mechanisms of sporangial emptying represent steps in an evolutionary transition series. We have reevaluated this idea in a phylogenetic framework using internal transcribed spacer sequences of four genera. These data were compared with the response to osmotic stress exhibited by each taxon. Saprolegnia emerges as the most basal genus, sister to Achlya, Thraustotheca, and Dictyuchus. Achlya and Thraustotheca are most closely related, while Dictyuchus appears to have evolved along a separate evolutionary lineage. The resulting phylogenetic framework is consistent with the idea that the mechanism of sporangial emptying exhibited by Saprolegnia represents the plesiomorphic condition from which the other mechanisms were derived independently. These alternative mechanisms of spore release may have resulted from a small number of mutations that inhibited axonemal development and altered the temporal and spatial expression of lytic enzymes that degrade the sporangial wall. Copyright 1998 Academic Press.     

Targeted Inactivation of Mouse RAD52 Reduces Homologous Recombination but Not Resistance to Ionizing Radiation

The RAD52 epistasis group is required for recombinational repair of double-strand breaks (DSBs) and shows strong evolutionary conservation. In Saccharomyces cerevisiae, RAD52 is one of the key members in this pathway. Strains with mutations in this gene show strong hypersensitivity to DNA-damaging agents and defects in recombination. Inactivation of the mouse homologue of RAD52 in embryonic stem (ES) cells resulted in a reduced frequency of homologous recombination. Unlike the yeast Scrad52 mutant, MmRAD52^−/− ES cells were not hypersensitive to agents that induce DSBs. MmRAD52 null mutant mice showed no abnormalities in viability, fertility, and the immune system. These results show that, as in S. cerevisiae, MmRAD52 is involved in recombination, although the repair of DNA damage is not affected upon inactivation, indicating that MmRAD52 may be involved in certain types of DSB repair processes and not in others. The effect of inactivating MmRAD52 suggests the presence of genes functionally related to MmRAD52, which can partly compensate for the absence of MmRad52 protein.     

Molecular dosimetry of DNA damage caused by alkylation. II. The induction and repair of different classes of single-strand breaks in cultured mammalian cells treated with ethylating agents

Cultured Chinese hamster ovary cells were treated with ethylating agents. DNA lesions giving rise to single-strand breaks (SSB) or alkali-labile sites were measured by elution through membrane filters at pH 12.0 and pH 12.6, and by centrifugation in alkaline sucrose gradients after 1 h and 21 h lysis in alkali. Two agents with different tendencies to ethylate preferentially either at N or O atoms were compared, namely N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG) and diethyl sulphate (DES). The compounds differed greatly in their potency to induce lesions, but the ratios of SSB, measured with different methods after a treatment for 30 min, did not differ significantly. This suggested that the spectrum of lesions induced by the two compounds is very similar. However, when both agents were studied with alkaline elution at pH 12.0 after a short treatment time (5 min) only ENNG was found to induce rapidly-repairable SSB. Most of these were rejoined already within 5 min after treatment. These results suggest that rapidly-repairable lesions occurring in DNA after treatment of mammalian cells with ethylating agents are due mainly to alkylation at O-atoms.     

Phylogeography of the magpie-robin species complex (Aves: Turdidae: Copsychus) reveals a Philippine species, an interesting isolating barrier and unusual dispersal patterns in the Indian Ocean and Southeast Asia

Aim The oriental magpie‐robin (Copsychus saularis) of South and Southeast Asia is a phenotypically variable species that appears to be closely related to two endemic species of the western Indian Ocean: the Madagascar magpie‐robin (Copsychus albospecularis) and the Seychelles magpie‐robin (Copsychus sechellarum). This unusual distribution led us to examine evolutionary relationships in magpie‐robins, and also the taxonomic significance of their plumage variation, via a molecular phylogenetic and population genetic analysis of C. saularis and C. albospecularis. Location Southern Asia from Nepal across Indochina to southern China, and the Indian Ocean from Madagascar to the Greater Sunda and Philippine islands. Methods We sequenced 1695 nucleotides of mitochondrial DNA comprising the complete second subunit of the nicotinamide adenine dinucleotide dehydrogenase (ND2) gene and 654 bases of the cytochrome c oxidase subunit I (COI) region in 51 individuals of eight C. saularis subspecies, 10 individuals of C. albospecularis (one subspecies) and single individuals of two other Copsychus species as outgroups. The data were analysed phylogenetically, with maximum likelihood, Bayesian, relaxed clock and parsimony methods, and geographically for patterns of genetic diversity. Results Phylogenetic analysis indicated that C. albospecularis lies within the nominal C. saularis, making C. saularis polyphyletic. Malagasy and non‐Philippine Asian populations form a monophyletic group that is sister to a clade of Philippine populations. Within non‐Philippine Asian populations, two groups are evident: black‐bellied birds in the eastern Greater Sunda islands and white‐bellied birds in the western Sundas and on mainland Asia. Main conclusions The phylogeny of magpie‐robins suggests a novel pattern of dispersal and differentiation in the Old World. Ancestral magpie‐robins appear to have spread widely among islands of the Indian Ocean in the Pliocene, probably aided by their affinity for coastal habitats. Populations subsequently became isolated in island groups, notably the Philippines, Madagascar and the Greater Sundas, leading to speciation in all three areas. Isolation in the Philippines may have been aided by competitive exclusion of C. saularis from Palawan by a congener, the white‐vented shama (Copsychus niger). In the Greater Sundas, white‐bellied populations appear to have invaded Borneo and Java recently, where they hybridize with resident black‐bellied birds.     

Local people value environmental services provided by forested parks

Garnering support from local people is critical for maintaining ecologically viable and functional protected areas. However, empirical data illustrating local people’s awareness of the importance of nature’s services is limited; hence possibly impeding effective ecosystem (environmental)-services based conservation efforts. Using data from five protected forests in four developing Southeast Asian countries, we provide evidence that local people living near parks value a wide range of environmental services, including cultural, provisioning, and regulating services, provided by the forests. Local people with longer residency valued environmental services more. Educated as well as poor people valued forest ecosystem services more. Conservation education has some influence on people’s environmental awareness. For conservation endeavors to be successful, large-scale transmigration programs should be avoided and local people must be provided with alternative sustenance opportunities and basic education in addition to environmental outreach to reduce their reliance on protected forests and to enhance conservation support.